Streptolysin S targets the sodium-bicarbonate cotransporter NBCn1 to induce inflammation and cytotoxicity in human keratinocytes during Group A Streptococcal infection

Abstract

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomally-produced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo. We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-κB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections.

Co-PATHOgenex web application for assessing complex stress responses in pathogenic bacteria

“…Streptolysin S, a peptide toxin generated by GAS, can induce lysis of red blood cells in humans, thereby conferring an advantage to the pathogen through the utilization of hemoglobin as a source of iron ( 19 ). Although the cytolytic capabilities of this pathogen have been studied for a century, the triggers and post-transcriptional mechanisms of toxin production remain elusive ( 20 ). In this case study, we focused on the triggers of streptolysin S genes’ expression where we constructed a co-expression network with default settings ( Fig.…”

Streptococcal peptides and their roles in host-microbe interactions

“…For example, several studies have explored ways to inhibit SLS-mediated effects on host cells. Small molecule inhibitors of proteins involved in ion transport, such as DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonate) and the N-cyanosulfonamide S0859 have been used to inhibit the lysis of erythrocytes and keratinocytes in response to SLS ( Higashi et al., 2016 ; Hammers et al., 2022 ). Interestingly, S0859 treatment was also able to prevent SLS-mediated NF-κB activation in keratinocytes, indicating that small molecule inhibition of the toxin can also prevent the associated damaging pro-inflammatory responses ( Hammers et al., 2022 ).…”

“…SLS is most well-known for its ability to rapidly lyse erythrocytes, which was traditionally attributed to the ability of the toxin to disrupt host membranes ( Duncan and Mason, 1976 ; Nizet et al., 2000 ; Carr et al., 2001 ). However, recent studies have indicated that SLS lyses host cells by targeting membrane proteins that are associated with ion transport ( Higashi et al., 2016 ; Hammers et al., 2022 ), and several reports have expanded the role of SLS in the context of Streptococcal infections. The toxin is known to play an important role in invasive GAS infections ( Betschel et al., 1998 ; Datta et al., 2005 ; Hirose et al., 2019 ), and is able to disrupt multiple host cells in addition to erythrocytes.…”

“…Subsequent studies demonstrated that IL-1β was especially upregulated in an SLS-dependent manner during these infections ( Flaherty et al., 2018 ), providing additional support for the role of this cytokine in GAS infections ( Wang et al., 2008 ; Valderrama et al., 2017 ; Richter et al., 2021a ; Richter et al., 2021b ). SLS appears to induce these signaling events in keratinocytes by targeting the membrane protein NBCn1 in this context ( Hammers et al., 2022 ), thereby allowing GAS to promote disease severity by manipulating the host immune system and triggering excessive inflammation during infections. Exposure to SLS also triggers pro-inflammatory responses in host cells other than keratinocytes.…”

Streptolysin S is required for Streptococcus pyogenes nasopharyngeal and skin infection in HLA-transgenic mice

“…SLS also targets and disrupts the function of multiple cell types including erythrocytes, macrophages, neutrophils, and keratinocytes [11][12][13][14]. Specifically, SLS has recently been shown to interact with ion transporters on the cell surface of erythrocytes and keratinocytes to induce cytotoxicity [15,16]. Furthermore, SLS can cause the disruption of the epithelial barrier through the degradation of tight junctions in vitro [17].…”